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Journal of Agricultural Science and Technology B 2 (2012) 703-712 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Molecular Similarity between Gibberellic Acid and
Gliotoxin: Unravelling the Mechanism of Action for Plant
Growth Promotion by Trichoderma harzianum
Jaco Bezuidenhout1, Leon Van Rensburg1 and Peet Jansen van Rensburg2
1. School of Environmental Sciences and Development, North-West University, Potchefstroom Campus, Potchefstroom 2520, South
Africa
2. School of Physical and Chemical Sciences, North-West University, Potchefstroom Campus, Potchefstroom 2520, South Africa
Received: January 11, 2012 / Published: June 20, 2012.
Abstract: Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum include effects such as complete and even stand of plants, improved seed germination, increases in plant height and overall enhanced plant growth. No research has yet been conducted to elucidate the mechanism by which these effects occur. Improved seed germination, in particular, suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. The metabolite gliotoxin, produced by Trichoderma harzianum appear to be structurally most similar to gibberellic acid. In this study, common pharmacophore generation and molecular ligand docking simulations were used to evaluate the molecular similarity between gibberellic acid, specifically GA3, and gliotoxin. For the common pharmacophore evaluation, the structure of various gibberellic acids were used to construct a pharmacophore space to which gliotoxin was aligned, and during the molecular docking simulations the gibberellic acid receptor, GID1, served as ligand target for GA3 and gliotoxin. During the common pharmacophore evaluation, gliotoxin was successfully aligned to the common pharmacophore model constructed from various gibberellic acids. Furthermore, molecular docking simulations of gliotoxin and GA3 into the gibberellic acid receptor (GID1) yielded docking scores of -10.78 kcal/mol for the GA3 molecule from Corina and a docking score of -10.17 kcal/mol for gliotoxin. The docking scores suggest that gliotoxin may be able to competitively occupy the receptor space for gibberellic acid, and as such elicit the similar physiological responses observed in literature. Key words: Gibberellic acid, gliotoxin, GID1, molecular similarity, Trichoderma harzianum.
1. Introduction
As early as 1930, the potential of Trichoderma to
serve as a biological control agent was recognised and
research is increasing the list of diseases controlled by
this genus of fungus. This has lead to the commercial
production of several Trichoderma species and
Trichoderma-based products in countries such as
Israel, New Zealand, India, Sweden and South Africa
Leon van Rensburg, professor, research fields: plant physiology and ecological remediation. E-mail: [email protected].
Peet Jansen van Rensburg, M.Sc., research field: analytical biochemistry. E-mail: [email protected]. Corresponding author: Jaco Bezuidenhout, Ph.D., research field: plant protection. E-mail: [email protected].
for crop-protection and growth enhancement [1].
Literature reports that certain Trichoderma strains
are known to produce a variety of classes of bioactive
metabolites such antibiotics of the peptaibols class, as
well as inhibitors of fungal growth of a mainly
terpenic nature [2]. Overall, the production of
secondary metabolites in Trichoderma species is
strain dependent and includes volatile and non-volatile
antifungal substances such as
6-n-pentyl-6H-pyran-2-one (6PP or
6-pentyl-α-pyrone), gliotoxin, viridin, harziaopyridone,
harziandione and peptaboils [3]. Gliotoxin was first
described in 1934 and initially the compound was
D DAVID PUBLISHING
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
704
described as a “lethal principle” produced by
Trichoderma lignorum [4]. By 1941 this “lethal
principle” was characterised further and demonstrated
to be toxic to both R. solani and Sclerotinia
americana and named gliotoxin [1].
The mechanisms by which plants control growth
are many faceted and complex. One of these
mechanisms entails the so-called “plant growth
substances” or plant hormones [5]. According to Hill
[5], a plant growth substance can be defined as: “an
organic substance which is produced within a plant
and which will at low concentrations promote, inhibit
or qualitatively modify growth, usually at a site other
than its place of origin”. A further group of
compounds is possible with this definition as a base.
They are the “plant regulators” and they can be
defined as compounds whose effects, when applied to
plants, closely resemble that of the plant hormone. A
variety of these compounds are known and some of
them are chemical analogues of the endogenous plant
hormones, though not all.
Gibberellic acids (GAs) (also referred to as
gibberellins) are classified as tetracyclic diterpenoid
plant growth regulators. According to current studies
126 GAs have been identified in higher plants, fungi
and bacteria. Gibberellic acid regulates various
developmental and growth processes in plants such as
stimulation of seed germination; stimulation of stem
elongation; flowering; stimulation of pathenocarpy,
regulation of gene expression and stimulation of
trichome development. The action of gibberellic acid is
however antagonized by abscisic acid. Of the various
known gibberellins only a few are biologically active
in plants, these are GA1, GA3, GA4 and GA7 [6-10].
One particularly interesting phenomenon observed
when seeds or crops are treated with Trichoderma
species, is the complete and even stand of the treated
plots compared to the uneven and random stand of
untreated plots [1]. In a study of seed vigour of peas in
potting soil, Zheng and Shetty [11] also showed that
treatment with various Trichoderma species resulted
in increased and faster seed germination, increases in
plant height, an increase in phenolic compound
content in the seedlings and overall enhancement of
plant growth [11-13]. During this particular study
Trichoderma viride, Trichoderma harzianum and
Trichoderma pseudokoningii were compared. During
the particular study Trichoderma harzianum treatment
of the peas resulted in the highest average plant height
after 5 days, highest average weight of fresh seedlings
and highest phenolic compounds in the seedlings
relative to the other strains evaluated [11]. Another
study by Vinale et al. [13] also demonstrated that
secondary metabolites from Trichoderma have a role
in activation of plant defences as well as plant growth
regulation.
This suggests that treatment with Trichoderma
affect not only plant pathogens, but at the same time
the plant itself is also affected. Of all the mechanisms
employed by Trichoderma, the secretion of antibiotic
substances seems the most probable route. It is
suggested that some of the secondary metabolites
excreted by Trichoderma may function as a
homologue for a plant growth controlling substance or
hormone. In a review by Vinale et al. [13], studies are
cited where koninginin A and 6PP were evaluated for
plant growth promotion. At high concentrations (10-3
M) growth inhibitory effects were observed, however
at concentrations in the range of 10-5 M and 10-6 M
these compounds exhibited optimal auxin-like effects.
However, the auxin-like effects observed for
koninginin A and 6PP does not account for all the
effects observed from Trichoderma treatments. In
particular, the complete and even stand, but more
indicatively, the earlier germination of treated seeds
following Trichoderma treatment, suggests that the
effect of the plant hormone gibberellic acid, or
gibberellin, is being mimicked. Evaluation of the
various secondary metabolites secreted by
Trichoderma harzianum suggests that gliotoxin is the
most likely candidate to elicit these physiological
responses in plant systems.
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
705
Tools such as X-ray crystallography, nuclear
magnetic resonance and computational chemistry and
modelling are providing researchers with valuable
data to design and study ligand/substrate and protein
interactions in the fields of chemistry, biochemistry
and pharmacology [14]. Parallel to this, there has been
a great increase in the number of high-resolution
protein structures deposited in the Brookhaven Protein
Databank (PDB). This has enabled successful drug
design and evaluation in particularly the field of
pharmacology [15]. In the field of computer aided
drug design, ligand-protein interactions are a useful
tool to design and evaluate potential ligands against a
protein of interest [16, 17].
Docking-ligand studies can be described as a
target-based method [18]. During docking, various
interactions between the ligand and the protein must
be considered such as shape complementarity,
charge-charge interactions, solvation-desolvation
interactions, hydrophobic interactions and hydrogen
bonding [14]. Potentially suitable ligands are usually
selected based on a molecular binding scoring
function [16]. In general, lower energy scores indicate
better protein-ligand bindings when compared to
higher energy scores. As a result, in most cases the
docking is an attempt to optimise the computations to
find the lowest binding energy [19].
A pharmacophore represents a qualitative
prediction of binding by specifying the spatial
arrangement of a small number of atoms of functional
groups or in other words a 3D arrangement of atoms
or functional groups necessary to bind to a given
receptor [17, 18, 20]. Various critical interactions,
relatable to chemical features of the compound,
include hydrogen bonding, charge transfer, steric and
electrosteric properties, as well as lipophilic
interactions [18]. The advantage of using this
approach is that prediction and screening can be
performed on large databases as the pharmacophore
serves as a guide for searching for compounds or the
synthesis of new compounds and has been
successfully applied to a multitude of drug
development programs [17].
Due to the hydrophobic properties of gibberellic
acid, it has been postulated that gibberellic acid may
have both membrane-bound and soluble receptors in
plant cells [21]. Until recently research has as yet not
completely homed in on the specific receptors for
gibberellic acid, however the list of intracellular GA
signal transduction elements has been expanded to
include G-proteins and protein kinases [8]. However,
in the past decade various factors have been identified
through studies of rice (Oryza sativa) and Arabidopsis
mutants [22, 23]. Recently, Gibberellin-Insensitive
Dwarf1 (GID1) has been identified as a soluble
receptor for GA in both rice and Arabidopsis [21].
The GID1 proteins display a close structural
similarity to hormone sensitive lipases such as those
found in higher animals, being a globular protein and
containing a pocket for the substrate. Gibberellic acid
functions as an allosteric activator in GID1, allowing
structural changes that enable the receptor to associate
with DELLA proteins, however GA does not interact
with DELLA proteins by itself [10, 24-26].
In this study molecular docking and common
pharmacophore evaluation has been applied to
evaluate possible molecular similarity between
gibberellic acid and gliotoxin.
2. Materials and Methods
2.1 Molecular Modelling and Docking
All the proteins used for this study were acquired
from the Research Collaboratory for Structural
Bioinformatics (RCSB) Protein Databank
(www.rcsb.org). The Protein Data Bank (pdb) file
3ED1 (Crystal structure of rice GID1 complexed with
GA3) was used in the docking studies for GID1 and
GA3 and gliotoxin [26]. The structure files for GT,
GA1, GA3, GA4, GA5, GA7 and GA8 created online
using CORINA [27] from its SMILES (simplified
molecular-input line-entry specification) notation and
saved as a pdb file.
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
706
2.2 Molecular Similarity
The molecular similarity evaluation was only
conducted between gliotoxin and GA3, the primary
molecules of interest in this study. Common feature
pharmacophore generation using the HipHop
algorithm of the Catalyst molecular modelling
software suite version 4.8 [28] was applied to evaluate
similarity between the molecules. During the
pharmacophore model generation gliotoxin was tested
against a model generated by using GA1, GA3, GA4,
GA5, GA7 and GA8 as input.
2.3 Molecular Docking
For molecular docking the pdb file 3ED1 (Crystal
structure of rice GID1 complexed with GA3) served as
the target with GA3 and gliotoxin serving as ligands.
The software package ArgusLab [29] was used for the
molecular docking and scorings. The ArgusDock
exhaustive search docking engine was used with a grid
resolution of 0.25 Ängstrom, docking precision set to
high precision and flexible ligand docking mode
enabled.
3. Results and Discussion
3.1 Common Pharmacophore Modelling
For reference purposes the structures of gibberellic
acid (GA3) and gliotoxin (GT) is presented in Fig. 1a
and Fig. 1b respectively.
The common pharmacophore model generated from
the various gibberellic acid molecules identified three
region types of interest for the pharmacophore
alignment, namely the hydrogen bond donor regions
(represented as magenta), hydrophobic regions
(represented as light blue, and hydrogen acceptor
regions (represented as green). When aligning the GA3
into this pharmacophore model it was observed that a
hydrophobic region (light blue) is located to the C2
and C3 side of the structure and was situated in close
proximity to the lactone ring between C4 and C10.
Hydrogen bond acceptor areas (green) lie between C9
and C10, as well as C10 through C11. In relative close
proximity to this, the hydrogen bond donor areas
(magenta) are located in the vicinity of C13 through
C14 (Fig. 2).
As is apparent from Fig. 3, gliotoxin was observed
to successfully align in the common pharmacophore
model that has been generated with the hydrogen bond
acceptor regions (green) being located in the
proximity of the carboxyamide group and C12, while
the hydrogen bond donor regions (magenta) were
located in the proximity of C1 to C2 in relative close
proximity the disulphide bridge. The hydrophobic
region (light blue) was observed to align in the
proximity of C7 to C8.
The structural analysis of GA3 and GT separately
within the common pharmacophore model suggests
that GT and GA3 might be perceived as being similar
in a plant system. This perception in plant systems is
significantly strengthened when the structures are
overlaid (Fig. 4) onto each other within the
pharmacophore model. The lactone ring of GA3
overlays with the ring structure at C5 for GT, while
the disulphide bridge at C1 and C11 overlays with the
ring structure at C8-C13 in GA3 (Refer to Fig. 1 for
numbering scheme employed). The spatial occupation
(a) (b)
Fig. 1 Numbered structures for gibberellic acid 3 (a) and gliotoxin (b).
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
707
(a) (b)
Fig. 2 Structure of GA3 as aligned to the common pharmacophore model. (Colour coding employed by CATALYST:
Magenta = hydrogen bond donor, Light Blue = hydrophobic region, Green = hydrogen bond acceptor).
(a) (b)
Fig. 3 Structure of gliotoxin as aligned to the common pharmacophore model. (Colour coding employed by CATALYST:
Magenta = hydrogen bond donor, Light Blue = hydrophobic region, Green = hydrogen bond acceptor).
(a) (b)
Fig. 4 Stick representation of GA3 (red) and gliotoxin (green) as aligned to common pharmacophore model.
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
708
of the molecules was also observed to be strikingly
similar. Thus, from common pharmacophore modelling,
the match between the molecules appear quite
convincing, but it is important to remember that during
common pharmacophore modelling only the functional
characteristics are considered for model generation and
alignment. In essence, gliotoxin should be able to bind
to the same receptors as GA3 does based on functional
group interactions, but this does not guarantee that
similar plant responses may be evoked.
3.2 Docking Simulation of GA3(gibberellic acid 3)
and GT (gliotoxin) into GID1
For reference purposes the structural basis of
gibberellin (GA3)-induced DELLA recognition by the
gibberellin receptor (GID1) from Shimada et al. [26]
are presented first (Protein file 3ED1 from RCSB).
The crystallised structure of GA3 is shown in Fig. 5a
and 5b.
As stated in the study by Shimada et al. [26] the
following apparent features were observed:
Arg244 and Ser116 and Ser191 and its orientation
relative to the carboxylate group.
Ile126, Val239 and Val319 (Leu323 not included in
the binding site defined by ArgusLab) and its
orientation relative to the aliphatic rings of GA3.
Ile24, Phe27 and Tyr31 (His119 not included in the
binding site defined by ArgusLab) are the N-terminal
extension helices which are adjusted for GA ring
recognition.
Tyr127 bond with the C3 hydroxyl group of GA3.
Phe238 form a weak bond with C13 hydroxyl group
of GA3.
For further comparison of the possible structural
similarity, a docking simulation was performed in
ArgusLab, using a GA3 molecule constructed from
SMILES notation and optimised using CORINA.
The docking pose for the optimised GA3 molecule
obtained with CORINA was observed to be situated
relatively higher than that for the GA3 molecule
crystallised within 3ED1 (Fig. 6 a and b as well as Fig.
7a and b). The orientation of the carboxylate group
was similar. However, the orientation in terms of the
lactone ring and hydroxyl groups appears to be flipped.
A docking score of -10.78 kcal/mol was the optimal
pose calculated. The change in the docking pose might
be a result of the GA3 from CORINA having a more
sterical hindrance than that of the GA3 molecule
within 3ED1. The differences in the optimal pose
calculated may also be due to differences between the
optimal configuration calculated for the generated
GA3 and the structure of the GA3 molecule within the
receptor protein, a factor CORINA does not account
for in the optimisation of the structure.
(a) (b)
Fig. 5 GA3 binding within the binding site of 3ED1. Stick representation (a), and the sphere representation (b).
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
709
(a) (b)
Fig. 6 GA3 (Generated with CORINA) binding within the binding site of 3ED1. Stick representation (a) and sphere representation (b).
(a) (b)
Fig. 7 Comparison of the docking poses for the GA3 molecule crystallised with 3ED1 (blue) and the GA3 molecule from CORINA. Docking poses with the binding site displayed (a) and docking poses with the binding site removed (b) for greater clarity. In Fig. 7b, the GA3 molecule within 3ED1 is represented as wireframe and the GA3 from CORINA is represented by a stick representation.
(a) (b)
Fig. 8 GT (Generated with CORINA) binding within the binding site of 3ED1. Stick representation (a) and sphere representation (b).
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
710
(a) (b)
Fig. 9 Comparison of the docking poses for the GA3 molecule crystallised with 3ED1 (red) and the GT molecule from CORINA (green). Docking poses with the binding site displayed (a) and docking poses with binding site not displayed (b) for greater clarity. In Fig. 9b, the GA3 molecule from CORINA is represented as wireframe and the GT from Corina is represented by a stick representation.
(a) (b)
Fig. 10 Comparison of the docking poses for the GA3 molecule crystallised with 3ED1 (blue) and the GT molecule from CORINA (green). Docking poses with the binding site displayed (a) and docking poses with binding site not displayed (b) for greater clarity. The GA3 molecule within 3ED1 (Fig. 10 b) is represented as wireframe and the GT from CORINA is represented as a stick representation.
When comparing the docking of gliotoxin and the
GA3 molecule crystallised into the structure of GID1,
the results appear to be more in line with the
orientation calculated from pharmacophore generation.
When the docking poses of both GA3 (from the GID1
structure itself) and GT are examined, the docking
pose for GT appears very close to that of the GA3
within the GID1 protein structure (Fig. 10a). Also,
when the molecules’ docking poses were compared
without the docking cage (Fig. 10b), the orientation
seemed to match that of the results during the
common pharmacophore evaluation. In this docking,
the lactone ring of GA3 aligned with the ring structure
at C5 (Refer to Fig. 1), while the disulphide bridge at
C1 and C11 aligned with the ring structure at C8-C13
in GA3. Furthermore, the COH group at C1 of GT
aligned with the carboxylate group at C6 from GA3 as
well as with Arg244, Ser116 and Ser191 of the protein.
The disulphide bridge of GT orientated with Val319
and a resulting docking score of -10.18 kcal/mol, was
Molecular Similarity between Gibberellic Acid and Gliotoxin: Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum
711
calculated by ArgusLab for the optimal pose.
Overall, with GT docked within 3ED1, gliotoxin
does appear to dock in a pose that is quite similar to
that predicted by the common pharmacophore
alignment. The COH group at C1 of GT appear to
orientate in a similar manner to the carboxylate group
of GA3. Alignment to the other functional groups
appears similar to that predicted from common
pharmacophore alignment. Again, sterical hindrance
might play a role in the alignment not being an exact
overlay match. Another reason for the differences
between the docking results obtained with regard to
molecule poses and the common pharmacophore
poses, might be that with the common pharmacophore
modelling only the characteristics of the molecules are
considered and aligned, whereas with docking,
molecular charges and various interactions between
the ligand and the binding site are allowed to affect
the final calculated orientation. In terms of docking
with the aid of ArgusLab, a docking score of -10.78
kcal/mol for the GA3 molecule from CORINA and a
docking score of -10.17 kcal/mol for GT was
calculated. Since the docking scores are comparable
for both compounds, this suggests that gliotoxin may
compete with gibberellic acid for the binding site
within GID1 [16].
Though the docking scores suggest good similarity
between the molecules, differences in the structure
and shape of the GA3 within GID1 and the external
GA3 (constructed using CORINA), might contribute
to the differences in docking poses observed. Thus,
based on the data presented above and the docking
scores obtained, it seems reasonable to deduce that GT
will dock to the GID receptor protein and may elicit
physiological responses similar to that of the natural
GA3 molecule.
Even though similarities between docking poses
and docking scores may strongly suggest similar
affinities for the ligands to the receptor, it still does
not guarantee that similar physiological responses will
be evoked. The evaluation of the physiological
responses was studied separately.
4. Conclusions
The results from the common pharmacophore
evaluation and molecular docking suggests a high
likelihood that gliotoxin may be perceived as
gibberellic acid in plant systems, however, molecular
similarity is not a guarantee that similar physiological
responses will be elicited in plant systems. However,
the approach may be valuable to screen candidate
plant growth regulators prior to evaluating the
physiological responses in plant systems.
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